AlphaBeta is a secondary alpha factor of Bacillus subtilis. Alpha Beta- containing RNA polymerase recognizes the promoters of several genes that are expressed at the end of exponential growth and during "Heat Shock". The activity of alphaB is modulated by an unusual cascade of negative regulators which includes an anti-sigma protein (RsbW) that binds to alphaB and blocks its ability to associate with RNA polymerase. The regulators are encoded by three genes (rsbV, rsbW and rsbX) that are cotranscribed with the alphaB structural gene. We propose to determine the functional relationships between these regulators and their target(s), the mechanism by which the effect their control and the signals to which they respond. Immunological probes for alphaB and its regulators have been developed and will be used to monitor their abundance, potential change of state, and associations. Regulated alphaB-dependent transcription will be reconstituted in vitro and used to directly test the interactions among the regulators and their targets. The rsbV, W, X system will be reconstituted in various combinations and ratios in vivo by placing one or more of the dependent transcription and the state of the other regulators. Using inappropriate activation of a alpha-dependent reporter gene as a screening criteria, mutations in genes whose products influence the activity of the rsbV, W, X system will be isolated and analyzed to determined the nature of the signals to which RsbV, W, and X respond. Finally a transposon-generated collection of reporter gene fusions will be used to identify and characterize additional operons that are controlled by the alphaB system. Control of the activity of ancillary alpha factors is critical to normal cell growth. Inappropriate activation of alphaB leads to an immediate and lethal induction of its regulon. A particularly intriguing aspects of alphaB control is its regulation by anti sigma factor. Anti-alpha factors represent a "new class" of regulatory molecules in prokaryotes. Their mode of action, regulation and the signals to which they respond are unknown. Unraveling their properties will not only provide new insights for global gene control but may also define unforeseen targets for antimicrobial agents.